Targeted analgesic microsphere based on piezoelectric material as well as preparation method and application of targeted analgesic microsphere

Abstract

The invention provides a targeted analgesic microsphere based on a piezoelectric material as well as a preparation method and application of the targeted analgesic microsphere. The method comprises the following steps: grafting rabies virus glycoprotein 29 peptide onto barium titanate particles, preparing a BaTiO3-RVG solution, then putting composite hydrogel prepared from GelMA and HAMA into the BaTiO3-RVG solution, and carrying out vacuum freeze-drying, so as to obtain the analgesic microspheres. The obtained analgesic microspheres can significantly reduce sodium ion inflow in DRG cells and expression of Nav1.7 channels and pain-related molecules, show a good analgesic effect, can inhibit pain signal conduction by regulating and controlling voltage-gated sodium channels, and provide a new thought for osteoarthritis pain treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a targeted analgesic microsphere based on piezoelectric materials, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is a progressive joint disease. Its main pathological processes include cartilage degeneration, extracellular matrix loss, and an imbalance between anabolism and catabolism during cartilage repair. Pain, the main clinical symptom of this disease, is closely related to the abundant peripheral sensory neurons in the joint tissues, which play a crucial role in pain signal transmission.

[0003] Currently, there are various treatment options for osteoarthritis pain, including traditional drug therapy, nerve block therapy, and surgical treatment. While systemic administration (such as oral medications) is more convenient for patients, long-term use may lead to adverse reactions and drug resistance. Nerve block therapy can provide precise pain relief, but the effect is short-lived, and surgery carries risks. Therefore, the management of OA pain remains a major clinical challenge.

[0004] Electrical stimulation therapy, as a physical therapy method, has shown significant efficacy in the management of osteoarthritis pain. However, traditional electrical stimulation therapy has drawbacks such as invasiveness, risk of infection, electrode breakage, localized pain, inaccurate localization, and lack of targeted efficacy. Therefore, developing a new approach to overcome these shortcomings is urgently needed in this field. Summary of the Invention

[0005] In view of the shortcomings of the prior art, one of the objectives of the present invention is to provide a method for preparing targeted analgesic microspheres based on piezoelectric materials for treating osteoarthritis pain.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] A method for preparing targeted analgesic microspheres based on piezoelectric materials for treating osteoarthritis pain, the method comprising the following steps:

[0008] Rabies virus glycoprotein 29 peptide was grafted onto barium titanate particles and a BaTiO3-RVG solution was prepared. Then, a composite hydrogel made of GelMA and HAMA was placed in the aforementioned BaTiO3-RVG solution and freeze-dried under vacuum to obtain the analgesic microspheres.

[0009] Preferably, the barium titanate particles are obtained by heat-treating a mixed solution of barium hydroxide, tetra(trimethylammonium chloride)titanium, titanium butoxide, ethanol and ammonium hydroxide at 200°C for 48 hours, followed by filtration, washing and drying.

[0010] Preferably, when grafting rabies virus glycoprotein 29 peptide onto barium titanate particles, the rabies virus glycoprotein 29 peptide is dissolved in a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a volume ratio of 5:3, then aminated barium titanate is added, and the reaction is stirred in an ice bath for 24 hours, followed by purification by ultrafiltration.

[0011] Preferably, the BaTiO3-RVG solution is obtained by placing barium titanate particles grafted with rabies virus glycoprotein 29 peptide in phosphate buffer.

[0012] Preferably, the weight ratio of GelMA to HAMA in the composite hydrogel is 5:1.

[0013] Preferably, the composite hydrogel is prepared using microfluidic technology.

[0014] Preferably, in the microfluidic technology, the aqueous phase in the microfluidic system consists of 5 wt% GelMA, 1 wt% HAMA and 1 wt% photoinitiator, while the oil phase consists of 95 wt% paraffin and 5 wt% Span80.

[0015] Preferably, in the microfluidic technology, liquids of different phases are injected into the microfluidic device at an oil-water flow rate ratio of 1000 μL / min: 20 μL / min. After pre-gelling and collection, the mixture is rapidly frozen with dry ice and then transferred to a freezer at -80°C for overnight storage. After cross-linking with 405 nm ultraviolet light, the mixture is washed three times with acetone and deionized water to finally obtain the composite hydrogel.

[0016] Another objective of this invention is to provide analgesic microspheres prepared by the above method.

[0017] Another object of the present invention is to provide the use of the aforementioned analgesic microspheres in the preparation of a pharmaceutical agent for treating osteoarthritis pain.

[0018] The beneficial effects of this invention are:

[0019] This invention develops an analgesic microsphere capable of generating electrical pulses. Under ultrasound stimulation, this microsphere activates nerve endings in the dorsal root ganglion (DRG), leading to the slow inactivation of the Nav1.7 channel and a reduction in sodium ion concentration within the DRG, thereby effectively blocking pain signal transmission and alleviating osteoarthritis pain. Notably, this analgesic microsphere also improves the pathological structure of damaged joints, significantly increasing the content of the anabolic protein COL2 while reducing the expression of the catabolic protein MMP13. This indicates that the analgesic microsphere of this invention has a positive effect on articular cartilage repair. In vitro and in vivo experiments both show that the analgesic microsphere of this invention significantly reduces intracellular sodium ion inflow into DRG cells and the expression of analgesic-related molecules in the Nav1.7 channel. Simultaneously, the analgesic microsphere of this invention also significantly improves pain behavior in rats, demonstrating a good analgesic effect. In summary, the ultrasound-responsive piezoelectric analgesic microsphere of this invention can inhibit pain signal transmission by regulating voltage-gated sodium channels, providing a new approach for the treatment of osteoarthritis pain. Attached Figure Description

[0020] Figure 1 The following are the characterization results of the analgesic microspheres obtained in this invention: Part A is the SEM image of barium titanate, Part B is the TEM image of barium titanate, Part C is the TEM and corresponding SAED image of barium titanate nanoparticles, Part D is the HRTEM image of barium titanate nanoparticles, Part E is the elemental distribution spectrum of barium titanate nanoparticles, Part F is the XRD spectrum and magnified peaks (inset), Parts G and H are the particle size and potential results of barium titanate, Parts I and J are the 1H nuclear magnetic resonance (NMR) analysis (600MHz, D2O) results of HA, HAMA, gelatin, and GelMA, Part K is the optical microscopic image of hydrogel microspheres (GA / HA), Part L is the particle size result of hydrogel microspheres, Part M is the SEM image of hydrogel microspheres, Part N is the SEM image of analgesic microspheres (BaTiO3-RVG@GA / HA), and Parts O and P are the output voltage results of hydrogel microspheres and analgesic microspheres.

[0021] Figure 2 The results of the biocompatibility study of analgesic microspheres are as follows: Part A shows the live and dead cell staining analysis of DRG cells (control group, ultrasound group, GA / HA group, BaTiO3-RVG@GA / HA group, and US@BaTiO3-RVG@GA / HA group) under different treatment conditions, with the experiment lasting for 1, 3, and 5 days; Part B shows the scaffold staining analysis of DRG cells under different treatment conditions, with the experiment lasting for 1, 3, and 5 days; Part C shows the proliferation analysis of DRG cells under different treatment conditions, with the experiment lasting for 1, 3, and 5 days; and Part D shows the toxicity analysis of DRG cells under different treatment conditions, with the experiment lasting for 1, 3, and 5 days. (NS indicates no significant difference, n=3).

[0022] Figure 3 The results of the targeting ability of analgesic microspheres are shown in Figure A. Part A shows bright-field images of dorsal root ganglion cells (control group, barium titanate group, and BaTiO3-RVG group) co-cultured for 1 day under different conditions. The yellow arrows indicate targeted barium titanate nanoparticles. Part B shows images of the dorsal root ganglion cell skeleton and nuclei after 1 day of culture under different conditions. The yellow arrows indicate targeted barium titanate nanoparticles. Part C shows in vivo fluorescence imaging of rat knee joints, showing the effect after 1 day of culture under different conditions. The yellow arrows indicate targeted barium titanate nanoparticles.

[0023] Figure 4 To demonstrate the analgesic effect of analgesic microspheres in vitro, Part A shows immunofluorescence co-staining images of dorsal root ganglion (DRG) cells under different conditions: red represents the cytoskeleton, blue represents DAPI, and green represents Nav1.7. Part B shows Na+ fluorescence staining images of DRG cells under different conditions: blue represents DAPI, and green represents Na+. Parts C and D show immunofluorescence staining of DRG cells for TRPV1 and CGRP under different conditions: red represents the cytoskeleton, blue represents DAPI, and green represents TRPV1 or CGRP. Part EH shows statistical analysis of fluorescence intensity. Part I shows the results of Western blot detection of Nav1.7, TRPV1, and CGRP. Part JL shows statistical analysis of protein expression levels. (NS indicates no statistical difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3).

[0024] Figure 5 To demonstrate the analgesic effect of analgesic microspheres in live animals, Part A shows the experimental design for rats; Part B shows the flowchart for the pain behavior test; Part C shows the trajectory and heat map of the open field test at different time points; Part D shows the changes in the footprint area of ​​the left and right hind limbs at different time periods; Part E shows the statistical analysis results of the open field test; Part F shows the statistical results of the gait analysis; and Parts G and H show the data of mechanical hyperalgesia and thermal hyperalgesia tests in rats at different time periods (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3).

[0025] Figure 6To illustrate the analgesic effect of analgesic microspheres in in vivo experiments, Part A presents the trajectory and thermograms of rats in different treatment groups (control group, MIA group, ultrasound group, GA / HA group, BaTiO3-RVG@GA / HA group, and US@BaTiO3-RVG@GA / HA group) in open field tests. Part B compares the hind paw footprint areas of rats in different treatment groups. Parts C and D present the statistical results of open field tests and gait analysis. Parts E and F present the mechanical hyperalgesia and thermal hyperalgesia tests of rats in different treatment groups. (NS indicates no statistical difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3).

[0026] Figure 7 The diagram shows the mechanism of action of analgesic microspheres in relieving pain. Part A compares the number of differentially expressed genes (DEGs) among the groups; Parts B and C show the DEG volcano plot analysis among the groups; Part D shows the GO enrichment analysis of DEGs in the MIA group and the control group; Part E shows the GO enrichment analysis of DEGs in the US@BaTiO3-RVG@GA / HA group and the MIA group; Part F shows the GSEA results of the inflammatory response; and Part G shows the GSEA results of sodium ion transmembrane transport.

[0027] Figure 8 Histological analysis of analgesic microspheres: Part A is hematoxylin-eosin staining; Part B is naphthol O-fast green staining; Part C is immunohistochemical images of COL2 and MMP13 in animal articular cartilage; Part D is the Mankin score of articular cartilage; Parts E and F are quantitative analysis of COL2-positive and MMP13-positive areas in cartilage (NS indicates no statistical difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3);

[0028] Figure 9 Figure 1 shows the characterization results of the preparation and function of analgesic microspheres. Part A shows the aqueous phase composition, Part B shows the oil phase composition, Part C shows the optical microscopic image of the microspheres (GA / HA), Part D shows the optical microscopic image of the analgesic microspheres (BaTiO3-RVG@GA / HA), Part E shows the elemental distribution spectrum of BaTiO3-RVG@GA / HA, Part F shows the drug loading efficiency of BaTiO3-RVG in the analgesic microspheres, Part G shows the thermogravimetric analysis curve of BaTiO3-RVG, Part H shows the Fourier transform infrared spectrum, Part I shows the fluorescence intensity comparison of released nanoparticles under different ultrasonic powers, Part J shows the percentage of released nanoparticles under different ultrasonic powers (NS indicates no statistical difference), and Part K shows the comparison of the piezoelectric constant d33 of barium titanate nanoparticles and analgesic microspheres.

[0029] Figure 10 Optical microscopic images of analgesic microspheres at different time points (see Part A), and fluorescence images of rat knee joints at different time points in vivo (see Part B);

[0030] Figure 11 The image shows the results of the immunofluorescence experiment. Part A shows the immunofluorescence staining of the rat knee joint under different conditions. Green represents nanoparticles, red represents CGRP, and blue represents DAPI. Part B shows the statistical analysis of fluorescence intensity (p<0.0001).

[0031] Figure 12 Immunofluorescence co-staining images of DRG cells under different conditions; red indicates the cytoskeleton, blue indicates DAPI, and green indicates Nav1.7.

[0032] Figure 13 The figure shows the results of assessing sodium levels in DRG cells under different conditions using ICP-MS (NS indicates no statistical difference, ****p<0.0001);

[0033] Figure 14 The figure shows the test results of mechanical hyperalgesia and thermal hyperalgesia in rats according to the present invention. Parts A and B show the test results of different doses of analgesic microspheres on mechanical hyperalgesia and thermal hyperalgesia in rats, and parts C and D show the test results of different ultrasound powers on mechanical hyperalgesia and thermal hyperalgesia in rats. (NS indicates no statistical difference, **p<0.01, ****p<0.0001).

[0034] Figure 15 The image shows the KEGG enrichment analysis results of this invention. Part A shows the KEGG enrichment analysis of differentially expressed genes between the MIA group and the control group, and Part B shows the KEGG enrichment analysis of differentially expressed genes between the US@BaTiO3-RVG@GA / HA group and the MIA group.

[0035] Figure 16 Images of the knee joint under different conditions. Detailed Implementation

[0036] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are still within the scope of protection of the present invention.

[0037] Example 1

[0038] 1. Preparation of BaTiO3:

[0039] A mixed solution consisting of 1.58 g of barium hydroxide (Ba(OH)₂·H₂O, 98%, Sigma-Aldrich), 2.27 g of tetrakis(trimethylammonium chloride)titanium (Ti[O(CH₂)₃CH₃]₄, 97%, Sigma-Aldrich), 1.62 g of titanium butoxide, 40 mL of ethanol, and 6 mL of ammonium hydroxide solution (30%, Sigma-Aldrich) was transferred to a sealed high-pressure reactor lined with polytetrafluoroethylene and heat-treated at 200 °C for 48 hours. After the reaction was completed, purified BaTiO₃ was obtained by filtration, washing, and drying.

[0040] 2. Synthesis of BaTiO3-RVG:

[0041] 1 mg of rabies virus glycoprotein 29 peptide (RVG) was dissolved in 2 mL of a 5:3 mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, Shanghai McKinron Biotech, China) and N-hydroxysuccinimide (NHS, Shanghai McKinron Biotech, China). Aminated barium titanate was then added, and the mixture was stirred in an ice bath for 24 hours. The product was purified by ultrafiltration and then dissolved in 2 mL of phosphate-buffered saline (PBS).

[0042] 3. Synthesis of GelMA and HAMA hydrogels

[0043] 10 g of gelatin (Gel, Shanghai Mailin Company) was completely dissolved in 100 mL of carbonate buffer (pH = 9.0) and reacted in an oil bath at 50 °C. Then, 2 mL of methyl acrylate (MA, Shanghai Aladdin Company) was slowly injected at a rate of 0.2 mL / min using a microinjection device, with continuous stirring for 3 hours to ensure complete reaction. Finally, 50 mL of PBS was added to terminate the reaction. Unreacted MA was removed by centrifugation (7000 rpm, 15 min, 25 °C), and the GelMA was dialyzed in deionized water (37 °C, 2 days). After lyophilization, the GelMA was stored at -20 °C. For HAMA, 10 g of hyaluronic acid (HA, Shanghai Mailin Company) was dissolved in 500 mL of water and reacted with 20 mL of MA at 0 °C and pH = 8-8.5 for 12 hours. After purification and lyophilization, the HAMA was stored at -20 °C.

[0044] 4. Synthesis of analgesic microspheres

[0045] In the microfluidic system, the aqueous phase consisted of 5 wt% GelMA, 1 wt% HAMA, and 1 wt% photoinitiator (Engineering Life Sciences, Suzhou, China), while the oil phase consisted of 95 wt% paraffin (McKinlun, Shanghai, China) and 5 wt% Span80 (Yuanye, Shanghai, China). Liquids of different phases were injected into the microfluidic device at a suitable oil-water flow rate ratio of 1000 μL / min:20 μL / min. After pre-gelling, the collected material was rapidly frozen with dry ice and then transferred to a -80°C freezer for overnight storage. After crosslinking with 405 nm UV light, the microspheres were washed three times with acetone and deionized water to obtain GA / HA microspheres. The GA / HA microspheres were then freeze-dried under vacuum, immersed overnight in BaTiO3-RVG solution, and freeze-dried again to obtain the final analgesic microspheres.

[0046] Experimental Example 1

[0047] 1. Characterization of BaTiO3:

[0048] The morphology, elemental distribution, and average diameter of barium titanate were analyzed using scanning electron microscopy (SEM, Czech Republic TESCAN CLARA), transmission electron microscopy (TEM, USA FEI G2 F20), and dynamic light scattering (DLS, UK Malvern Instruments Mastersizer 2000). X-ray diffraction (XRD, Germany Bruker D8 ADVANCE) was used to resolve the crystal structure.

[0049] Tetragonal barium titanate nanoparticles (BaTiO3) were prepared via hydrothermal synthesis. A typical tetragonal phase structure was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 1 Parts A and B). Selected area electron diffraction (SAED) analysis revealed that the barium titanate nanoparticles were defect-free, corresponding to the (001) and (010) crystal planes. Figure 1 (Part C). High-resolution transmission electron microscopy (HRTEM) shows interplanar spacings of 0.39 and 0.37 nm, respectively. Figure 1 Part D). Barium (Ba), titanium (Ti), and oxygen (O) elements are evenly distributed. Figure 1 Part E). X-ray diffraction (XRD) detected a double-peak diffraction feature at 2θ = 45°, which is a typical feature of the tetragonal piezoelectric material barium titanate. Figure 1 (Part F in the middle section). Dynamic light scattering (DLS) measurements showed that the average particle size of barium titanate was 92.89 nm, and the surface potential was -23.62 mV. Figure 1 (Parts G and H).

[0050] 2. Characterization of BaTiO3-RVG

[0051] The loading of RVG on the barium titanate surface was analyzed using Fourier transform infrared spectroscopy (FTIR, Nicolet 6700, USA). Finally, the relative contents of RVG and barium titanate in BaTiO3-RVG were determined by thermogravimetric analysis (TGA, Netzsch STA449F3, Germany).

[0052] The characteristic peak of RVG can be observed in the FTIR spectrum of BaTiO3-RVG, with the peak at 3396.5 cm⁻¹. -1 and 1083.8cm -1 The vibrational bands at the point correspond to the free amino and C=O groups in RVG, confirming that RVG has been successfully modified onto the surface of barium titanate.

[0053] Thermogravimetric analysis of BaTiO3-RVG showed that RVG began to decompose within the temperature range of 100-200℃, leading to a rapid mass decrease of 9.29%. Figure 9 (G part), this result strongly proves that the barium titanate surface modification was successful.

[0054] 3. Characterization of GelMA and HAMA hydrogels

[0055] The percentage of methacrylation of GelMA and HAMA was analyzed by proton nuclear magnetic resonance (1H NMR, JNEJT JNM-ECZ600R).

[0056] Hydrogen nuclear magnetic resonance (1H NMR) confirmed that methacrylic anhydride successfully modified hydroxyapatite (HA) and gel ( Figure 1 Parts I and J). To improve the injectability of the hydrogel, microfluidic technology was used to inject the aqueous phase (5% GelMA and 1% HAMA) into the hydrogel. Figure 9 Part A) and the oil phase (paraffin oil and Span 80) Figure 9 Part B) was made into W / O type spherical droplets, which were then cross-linked with ultraviolet light at -40℃ to prepare porous GA / HA microspheres.

[0057] 4. Characterization of analgesic microspheres

[0058] The morphology of the analgesic microspheres was observed using optical microscopy and scanning electron microscopy, and Fourier transform infrared spectroscopy was used for analysis. The drug loading efficiency of BaTiO3-RVG at different time points was obtained by detecting the mass change of the hydrogel microspheres before and after drug loading. The controlled release effect of the analgesic microspheres under different ultrasonic powers was measured using a fluorescence spectrophotometer (F-4700, Hitachi, Japan) and an enzyme-linked immunosorbent assay (INFINITE 200PRO, Tecumseh, Switzerland). Finally, the degradation and clearance of the analgesic microspheres at different time points were observed using optical microscopy and an in vivo fluorescence imaging system (IVScpoe 7550, CLINX Scientific Instruments Co., Ltd., Shanghai, China).

[0059] Optical microscopy revealed that the GA / HA microspheres were uniformly dispersed and had intact morphology. Figure 1 Part K of China Figure 9 The average particle size of the middle C portion is 247.81 ± 10.35 μm. Figure 1 (Part L in the middle). SEM analysis used to detect the surface morphology of GA / HA microspheres showed that the vacuum-dried microspheres have a loose porous structure, which is crucial for the effective loading of piezoelectric nanoparticles. Figure 1 (Middle M section).

[0060] Magnified SEM images and elemental composition analysis of BaTiO3-RVG@GA / HA confirmed that BaTiO3-RVG was effectively loaded into GA / HA microspheres. Figure 1 Part N Figure 9 (Part E). Drug loading efficiency analysis showed that the maximum drug loading rate of BaTiO3-RVG on GA / HA hydrogel microspheres was 16.6% ( Figure 9 (Part F of the middle section).

[0061] FTIR results for GA / HA showed 1643.5 cm. -1 The amide bond absorption peak at 3443.7 cm⁻¹ -1 The amino absorption peaks at the [specific locations] correspond to the characteristics of GelMA and HAMA, respectively, further verifying the modification effect. Consistent with the scanning electron microscopy (SEM) results, Fourier transform infrared (FTIR) spectroscopy analysis of BaTiO3-RVG@GA / HA showed that its characteristic peaks were in complete agreement with the BaTiO3-RVG and GA / HA materials described above. Figure 9 (H portion). These results confirm the successful synthesis of BaTiO3-RVG@GA / HA analgesic microspheres.

[0062] Controlled-release experimental data showed that the release of BaTiO3-RVG reached its peak when the ultrasonic power was 0.5 watts, and the release no longer increased with further increases in power. Figure 9 (Parts I and J).

[0063] Light microscopy observation results ( Figure 10 Part A) and in vivo fluorescence imaging data at different time points ( Figure 10 Part B shows that these analgesic microspheres gradually degrade and are eventually eliminated over time.

[0064] 5. Piezoelectric properties of analgesic microspheres

[0065] The piezoelectric constant d33 of barium titanate and BaTiO3-RVG@GA / HA was determined using a d33 measuring instrument (model ZJ-3A, Beijing, China). The output voltage of the microspheres under ultrasonic irradiation was tested using a voltage oscilloscope (model DSOX6004A, Keysight Technologies, USA). The microspheres were placed in deionized water and fixed to the ultrasonic probe, with the oscilloscope electrodes placed on the surface of the microspheres. The output voltage on the surface of the microspheres was measured and recorded under continuous wave and pulsed wave ultrasound (1.0 MHz, 5 minutes) at different powers (0.5 W, 1 W, 2 W, 3 W, 4 W).

[0066] Researchers measured the piezoelectric constant d33, which characterizes the piezoelectric effect. Experimental data showed that the piezoelectric constant of the BaTiO3-RVG@GA / HA material is approximately 11.7 picocoulombs / Newton, indicating that this analgesic microsphere possesses excellent piezoelectric properties. Figure 9 (Middle K section). Oscilloscope measurements revealed that although the GA / HA material itself produces almost no output voltage, the analgesic microspheres generated output voltages of 127 mV and 147 mV under continuous ultrasound and pulsed ultrasound, respectively. Figure 1 (Part O). It is worth noting that as the ultrasound power increases, the output voltage of the analgesic microspheres also increases synchronously ( Figure 1 (Part P of the middle section).

[0067] 6. Cell Culture

[0068] DRG rat dorsal root ganglion neurons were provided by Otto Biotechnology Co., Ltd. (Shenzhen, China). Cells were cultured in DMEM (Gibco, USA) containing 10% fetal bovine serum and 1% penicillin-streptomycin solution at 37°C and 5% carbon dioxide. For in vitro experiments, cells were cultured at a rate of 2 × 10⁶ cells / year. 4 Inoculate 6-well plates at a density of 100 cells / well and group them according to the wells. Change the culture medium every two days.

[0069] 7. Biocompatibility of pain-relieving microspheres

[0070] Dorsal root ganglion (DRG) cells were co-cultured with analgesic microspheres in 6 cm culture dishes. The toxicity of the analgesic microspheres to DRGs was assessed by live / dead cell staining, cytoskeleton staining, CCK8 cell counting assay, and lactate dehydrogenase (LDH) test. Cell viability at 1, 3, and 5 days after co-culture was assessed using a live / dead cell staining kit (Beyotime Biotechnology, China). DRG cells were incubated in a mixture of calcein-acetoxymethyl (AM) and propidium iodide for 30 minutes, followed by observation using a confocal microscope (Nikon, Japan). After 1, 3, and 5 days of co-culture, DRG cells were fixed with 4% paraformaldehyde for half an hour, followed by permeabilization with 0.1% Triton X-100 solution for 15 minutes. To observe the cytoskeleton and nuclear structure, Alexa Fluor 594 phalloidin and 4′,6-diamidindo-2-phenylindole (DAPI) staining were performed, followed by observation using a confocal microscope. Cell proliferation and cytotoxicity were assessed using CCK-8 (Beyotime Biotech) and LDH assay kits (Beyotime Biotech) after 1, 3, and 5 days of co-culture.

[0071] As a novel injectable biomaterial, analgesic microspheres should possess good biocompatibility. Therefore, this study used live / dead cell staining, cytoskeleton staining, and CCK-8 and LDH assays to evaluate the effects of BaTiO3-RVG@GA / HA on DRG neuron activity. The experiment was divided into five groups: control group, US group, GA / HA group, BaTiO3-RVG@GA / HA group, and US@BaTiO3-RVG@GA / HA group. After co-culturing DRG cells with microspheres for 1, 3, and 5 days, cell proliferation, viability, and morphological characteristics were assessed using live / dead cell staining and cytoskeleton staining. The results showed that DRG cells adhered and grew on the surface of the analgesic microspheres, confirming that GA / HA is an effective carrier material. Compared with the control group, the number of cells on the surface of the microspheres gradually increased over time, eventually covering the entire surface of the microspheres, and no dead cells were observed in any group. Figure 2 (Part A). Cytoskeleton staining showed that the cells on the surface of the microspheres grew normally, had intact structure and good elasticity, and no significant changes were observed during culture. Figure 2 Part B). CCK-8 assays revealed no significant difference in DRG cell proliferation after co-culturing for 1, 3, and 5 days. Figure 2 Part C). LDH test results also confirmed that the analgesic microspheres did not exhibit significant biotoxicity. Figure 2 (Part D). In summary, the analgesic microspheres BaTiO3-RVG@GA / HA exhibit good biocompatibility, which is the basis for their effective analgesic effect within the joint cavity.

[0072] 8. Targeting ability of analgesic microspheres

[0073] To fluorescently label BaTiO3-RVG, the inventors conjugated fluorescein isothiocyanate (FITC) to the material. After overnight stirring, the mixture was centrifuged and washed with water to remove unreacted substances. FITC-labeled BaTiO3-RVG was added to DRG cell culture medium and co-cultured for one day. Its targeting effect on cells was observed using confocal microscopy. After co-culturing FITC-labeled BaTiO3-RVG with DRG cells for one day, the DRG cells were fixed and permeabilized. To observe the cytoskeleton and nuclear structure, Alexa Fluor 594 phalloidin and DAPI staining were used, and the relative positions of FITC-labeled cells and DRG cells were observed using confocal microscopy. To observe in vivo targeting ability, rats were anesthetized with sodium pentobarbital solution (0.3 mL / 100 g), and after shaving the hair around the knee joint, FITC-labeled BaTiO3-RVG was injected into the knee joint cavity. The following day, in vivo fluorescence imaging was performed using an IVScpoe 7550 system (CLINX Scientific Instruments Co., Ltd., Shanghai, China). Simultaneously, frozen sections of rat knee joints were extracted and immunofluorescence stained with the neuromarker calcitonin gene-related peptide (CGRP) to further confirm the biodistribution of the nanoparticles within the knee joint. Finally, these sections were observed using a Leica DM4B fluorescence microscope.

[0074] As the GA / HA microspheres gradually degraded, BaTiO3-RVG nanoparticles were released. To verify the targeting ability of the nanoparticles, we used FITC to fluorescently label barium titanate and BaTiO3-RVG, and then co-cultured the labeled nanoparticles with DRG cells. After one day of co-culture, bright-field imaging was performed using confocal microscopy. The results showed that, compared with unmodified nanoparticles, BaTiO3-RVG significantly targeted DRG cells (…). Figure 3 (Part A). To confirm whether the targeting site of BaTiO3-RVG conformed to our hypothesis (DRG cell axons), we stained the nuclei and cytoskeleton of DRG cells. The results showed that BaTiO3-RVG effectively targeted DRG cell axons, while the barium titanate group showed no significant targeting ability. Figure 3 (Part B). In addition to verifying the cell-targeting ability of the nanoparticles, we also observed the in vivo targeting effect of BaTiO3-RVG using in vivo fluorescence imaging. After anesthetizing rats, FITC-labeled barium titanate or BaTiO3-RVG was injected into the right knee joint cavity. One day after injection, observation was performed using an in vivo fluorescence imaging system. The results of in vivo fluorescence imaging of the knee joint showed that the barium titanate nanoparticles were dispersed within the knee joint cavity, while the BaTiO3-RVG-modified nanoparticles targeted the synovium on both sides of the knee joint. Figure 3(Part C). This synovial targeting is significant because the knee synovium contains DRG nerve endings, which are the expected target sites for the nanoparticles. This result is consistent with in vitro experimental results, confirming that our biomaterials maintain effective targeting capabilities in complex physiological environments. Furthermore, immunofluorescence staining results showed that RVG-modified nanoparticles exhibited significantly increased enrichment in synovial nerves compared to unmodified nanoparticles. Figure 11 In conclusion, good targeting ability is the foundation for analgesic microspheres to exert their effects.

[0075] 9. Analgesic effect of in vitro analgesic microspheres

[0076] 20 ng of IL-1β was added to each experimental group except the control group to simulate an inflammatory environment. After one day of culture, different treatments were administered according to the groups: the ultrasound group and the US@BaTiO3-RVG@GA / HA group received 5 minutes of ultrasound stimulation (twice daily, frequency 1.0 MHz, power 0.5 W, duty cycle 50%, equipment provided by Chongqing Ronghai Ultrasonic Engineering Co., Ltd., China). 24 hours after ultrasound stimulation, 5 μM of the fluorescent sodium ion probe CoroNa Green (AM, Shanghai Maokang Biotechnology Co., Ltd.) was injected into the dorsal root ganglion cells, followed by observation using confocal microscopy. In addition, inductively coupled plasma mass spectrometry (ICP-MS) was used to further analyze the sodium ion level in the dorsal root ganglion cells. After treatment, the expression and distribution of Nav1.7, transient receptor potential vanillic acid isoform 1 (TRPV1), and calcitonin gene-related peptide (CGRP) were detected by immunofluorescence. After fixation and permeabilization, the cells were incubated with 1% bovine serum albumin (BSA) for 30 minutes. Subsequently, anti-Nav1.7 antibody (1:300, Proteintech, USA), anti-TRPV1 antibody (1:200, Proteintech, USA), or anti-CGRP antibody (1:300, ABclonal, China) were added. After incubation with the mixture, the cells were treated with fluorescently labeled secondary antibody (1:1000, Proteintech, USA) for 1 hour. After staining the cytoskeleton and nuclei, the cells were observed using a confocal microscope. Simultaneously, the protein expression levels of Nav1.7, TRPV1, and CGRP were verified by Western blotting. DRG cells receiving different interventions were collected and homogenized using a lysis buffer containing a protease inhibitor (Beyotime, China) and benzyl sulfonyl fluoride (PMSF) (Beyotime, China). After electrophoretic separation of proteins (30 μg), they were transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, USA). After blocking for 2 hours, the membranes were incubated with Nav1.7 antibody (1:1000 dilution, Proteintech, USA), TRPV1 antibody (1:1000 dilution, Abcam ab305299, UK), CGRP antibody (1:1000 dilution, Zhen Biotech, China), and β-actin antibody (1:5000 dilution, Proteintech, USA). Subsequently, the membranes were co-incubated with secondary antibody (1:5000 dilution, Proteintech, USA) for 2 hours. Finally, the bands were analyzed using a chemiluminescence immunoassay analyzer.

[0077] The analgesic microspheres developed in this invention generate electrical pulses through ultrasound, inactivating Nav1.7 channels and inhibiting sodium current generation, thereby blocking the transmission of pain signals to the central nervous system. This experiment aims to verify the analgesic effect of the analgesic microspheres in vitro. The inventors used IL-1β to simulate the in vitro inflammatory environment of osteoarthritis. The experimental groups included a control group, an IL-1β group, an ultrasound group, a GA / HA group, a BaTiO3-RVG@GA / HA group, and an ultrasound@BaTiO3-RVG@GA / HA group. Nav1.7 immunofluorescence results showed that Nav1.7 expression in dorsal root ganglion cells was significantly increased after IL-1β treatment, while the ultrasound@BaTiO3-RVG@GA / HA group significantly reversed this change. Figure 4 Parts A and E Figure 12 Furthermore, the inventors used a Na+ fluorescent label to label intracellular Na+ in dorsal root ganglion cells. The results showed that intracellular Na+ was significantly reduced in the ultrasound@BaTiO3-RVG@GA / HA group, while no significant changes were observed in other groups. Figure 4 (Parts B and F). ICP-MS can be used to analyze intracellular metal ion content. ICP-MS results showed that after applying ultrasound-responsive analgesic microspheres, the intracellular Na+ concentration was significantly reduced, further providing strong evidence for our findings. Figure 13 TRPV1 is expressed on nociceptive neurons and can be activated by various stimuli. In osteoarthritis (OA), nociceptive stimuli induced by joint inflammation and cartilage damage can activate TRPV1 receptors, thereby generating and transmitting pain signals. This invention validates the efficacy of analgesic microspheres by detecting pain-related TRPV1 molecules. Immunofluorescence results of TRPV1 showed that US@BaTiO3-RVG@GA / HA significantly reversed IL-1β-induced increases in TRPV1 expression. Figure 4 (C and G sections). Another type of pain-related peptide, CGRP, also exhibits similar effects. Figure 4 (Parts D and H). Pain stimuli induced by OA lead to increased CGRP levels, which in turn mediate pain signal transmission and exacerbate pain perception. These experimental results further confirm that the analgesic microspheres of the present invention exert their effects through the following mechanisms: regulating Nav1.7 channels; inhibiting Na+ ion influx to block pain signal transduction; and reducing the expression of pain-related TRPV1 and CGRP, thereby achieving effective analgesia. In addition to immunofluorescence detection, the inventors also used Western blotting to further verify the expression levels of these proteins, and the results were consistent with the immunofluorescence detection results. Compared with the prior art, US@BaTiO3-RVG@GA / HA can significantly reduce the expression of Nav1.7 channels and pain-related molecules TRPV1 and CGRP ( Figure 4(Middle IL portion). In summary, the analgesic microspheres of the present invention can not only directly inhibit Nav1.7 to regulate Na+ through electrical pulses, but also indirectly reduce the expression of pain signal transduction-related molecules, demonstrating excellent ability to relieve OA pain.

[0078] 10. Animal models and the analgesic effect of analgesic microspheres in vivo.

[0079] The experimental rats were Sprague-Dawley strain (weighing 180±20 g), provided by Chongqing Medical University. The rats were housed in a suitable environment (temperature 22±2°C, 12-hour light-dark cycle) with ample water and food. Five rats were housed per cage. This invention was approved by the Animal Ethics Committee of Chongqing Medical University (approval number: IACUC-CQMU-2024-07089). The rats were divided into 6 groups: control group, MIA group, US group, GA / HA group, BaTiO3-RVG@GA / HA group, and US@BaTiO3-RVG@GA / HA group. An OA pain model was used: after anesthesia, 60 μL of 80 mg / mL sodium iodoacetate monosodium salt (MIA, Sigma-Aldrich, USA) was injected into the right knee joint cavity. The injection site was the same for the control group. On day 22 after MIA injection, analgesic microspheres were injected into the right knee joint cavity.

[0080] All rats underwent intra-articular injection of microneedle arrays (MIA) in the right knee joint to establish an osteoarthritis pain model. Results regarding the relationship between analgesic microsphere dosage and pain-related behaviors showed that, compared to the 25 μL injection group, the three high-dose groups (50 μL, 75 μL, and 100 μL) had lower pain behavior thresholds (PWT). Figure 14 Part A) and pain threshold (PWL) Figure 14 All three groups showed significant improvement in aspects B (part of the study), but there was no significant difference among the three groups. Furthermore, the effect of ultrasound power on pain behavior indicated that the improvement was more pronounced when the power exceeded 0.5 watts, but the improvement ceased to be significant beyond 0.5 watts. Figure 14 (Parts C and D). Therefore, two 50 μL analgesic microsphere injections were administered on day 22 of the MIA injection, followed by 7 days of ultrasound irradiation (10 minutes / session / day, frequency 1.0 MHz, power 0.5 W, duty cycle 50%). Multiple behavioral tests, including open-field testing, gait analysis, mechanical hyperalgesia testing, and thermal hyperalgesia testing, were performed on the day before the MIA injection and on days 1, 3, 7, 14, 21, 24, 28, and 35 after the injection. Figure 5 Parts A and B). By observing behavioral changes in rats after MIA injection, the successful establishment of the OA pain model was confirmed. Open-field experiments showed that the total walking distance of rats after MIA injection significantly decreased and stabilized on days 14 and 21 post-injection. Figure 5(Parts C and E). Furthermore, hind limb footprints and footprint area were recorded through gait analysis. Results showed that after MIA injection, the footprint area of ​​the right hind limb significantly decreased, while the footprint area of ​​the left hind limb significantly increased, eventually reaching a stable state on day 21. Figure 5 (Parts D and F). The day after MIA injection, the range of motion in the right hind paw was significantly reduced, subsequently gradually recovering and stabilizing. The inventors speculate that this may be related to the acute inflammatory response induced by the MIA injection. Mechanical aberrations were assessed by measuring the passive pain threshold (PWT) using an electronic von Fryer sensor and detector, while thermal aberrations were detected by measuring the passive pain threshold (PWL) using a hot plate. The significant reduction in PWT and PWL after MIA administration compared to the control group confirms the successful establishment of the osteoarthritis pain model. Figure 5 (Parts G and H). Following MIA injection, OA pain in rats tended to stabilize after 21 days. 21 days after MIA injection, OA-affected rats were divided into 5 groups: MIA, US, GA / HA, BaTiO3-RVG@GA / HA, and US@BaTiO3-RVG@GA / HA. 22 days after MIA injection, analgesic microspheres were injected into the right knee joint cavity. Subsequently, the US treatment group received 7 days of treatment. After treatment, the total walking distance of the rats was observed through an open-field test. The results showed that the US@BaTiO3-RVG@GA / HA group significantly increased the total walking distance, while the other control groups showed no significant improvement. Figure 6 (Parts A and C). Multiple studies have shown that activity levels in rats are generally correlated with their pain state; pain often limits animal activity, leading to reduced mobility and exploratory behavior. Formalin-induced pain studies have shown that increased total walking distance may indicate reduced pain in rats. Furthermore, the US@BaTiO3-RVG@GA / HA group also exhibited a smaller footprint area on the right hind paw and an increased footprint area on the left hind paw. Figure 6 Parts B and D). Pain intensity and weight-bearing capacity were assessed through changes in gait analysis parameters. Studies have shown that reduced footprint area and decreased average contact intensity indicate pain, while increases in these indicators suggest better pain relief and enhanced weight-bearing capacity. Significant increases in PWT and PWL further confirm that US@BaTiO3-RVG@GA / HA can alleviate mechanical and thermal hyperalgesia. Figure 6(Parts E and F). Unlike other groups, the US@BaTiO3-RVG@GA / HA group showed a significant increase in PWT on day 24 after MIA injection. Notably, PWL also showed a significant improvement on day 28 after MIA injection. The differences in pain relief time induced by the analgesic microspheres measured in the tests may be related to the different fiber types targeted in each assessment. Thermal hyperalgesia is mainly mediated by C fibers and Aδ fibers, while mechanical hyperalgesia is mainly mediated by Aβ fibers. We found that on day 35 after MIA injection in rats, their pain threshold (PWT) and pain threshold (PWL) remained elevated, which may be related to the long-term inhibitory effect of the analgesic microspheres on pain transmission in rats, maintaining a high pain threshold level even after ultrasound treatment was stopped. In summary, these data indicate that US@BaTiO3-RVG@GA / HA plays a unique role in pain relief in osteoarthritis.

[0081] To assess pain behavior in rats, the inventors conducted open-field testing, gait analysis, mechanical hyperalgesia testing, and thermal hyperalgesia testing on days 1, 3, 7, 14, 21, 24, 28, and 35 post-MIA injection. All behavioral tests were conducted blinded, with observers unaware of group assignments beforehand. The optimal dosage was determined by observing the effects of different doses of analgesic microspheres (25 μL, 50 μL, 75 μL, and 100 μL) and ultrasound power (0 W, 0.1 W, 0.3 W, 0.5 W, 0.8 W, and 1 W) on pain behavior in rats. The analgesic microspheres were injected on day 22 post-MIA injection, followed by 7 days of ultrasound irradiation (Chongqing Ronghai Ultrasonic Engineering Co., Ltd., China). Open-field testing was conducted in a square testing device with a black frosted base, 50 cm high, with a base measuring 100 cm × 100 cm. Rats were placed in the center of the test area and allowed to explore freely for 5 minutes. Their activity trajectory was recorded and the total distance traveled (in meters) was calculated using a video tracking system (SA215, Shenzhen Sanan Biotechnology Co., Ltd., China). Gait analysis: A rodent gait fine analysis system (SA114, Shenzhen Sanan Biotechnology Co., Ltd., China) was used. Rats were placed at the open end of a glass-enclosed platform and allowed to move autonomously across the glass surface. Simultaneously, a high-speed camera below the platform captured images of the rats' paws, and the data was processed using gait analysis software. The inventors assessed the rats' pain level by measuring the largest footprint area (square centimeters). Mechanical hyperalgesia test: This was performed using an electronic von Fryer instrument (model 38450, Ugo Basilé, Italy). Specifically, each rat was individually housed in a cage with a mesh floor for one hour to allow acclimatization. Gradually increasing pressure was applied to the sole of the hind paw using a 0.5 mm diameter rigid polypropylene probe. The mechanical threshold was determined based on the pressure-induced foot retraction threshold (PWT), which was tested three times and the average value was recorded. Thermal hyperalgesia: 24 hours prior to the experiment, rats were placed on a hot plate (model SA707, Shenzhen Sansang Biotechnology Co., Ltd.) for 5 minutes to acclimatize. On the day of the test, the surface temperature of the hot plate was kept constant at 55±0.1℃. The rats' responses on the hot plate were closely observed, and the time when the first licking or jumping behavior appeared was recorded as the paw withdrawal latency (PWL).

[0082] 11. RNA-seq detection

[0083] The inventors used TRIzol reagent (Thermo Fisher Scientific, USA) to collect and purify total RNA from the control group, MIA group, and US@BaTiO3-RVG@GA / HA samples. This was then analyzed using Illumina NovaSeq. TMSequencing was performed using a 6000 sequencing platform (LC Biotechnology Co., Ltd., Hangzhou, China), and the high-quality sequencing data were analyzed using R language. Differentially expressed genes (DEGs) were identified based on the screening criteria of p-value ≤ 0.05 and |log2 (fold change)| > 1. These DEGs were further analyzed in depth using the Gene Ontology (GO) database and the Kyoto Encyclopedia of Genes and Genomes (KEGG).

[0084] To investigate the mechanism of action of analgesic microspheres in relieving pain, RNA sequencing analysis was performed on the control group, MIA group, and US@BaTiO3-RVG@GA / HA group. The results showed that 365 differentially expressed genes were found between the MIA group and the control group. Figure 7 Part A of the middle section). Volcano map analysis ( Figure 7 Part B revealed 48 upregulated genes and 317 downregulated genes, indicating that MIA induces multiple biological changes. On the other hand, 265 differentially expressed genes were observed between the US@BaTiO3-RVG@GA / HA group and the MIA group, including 130 upregulated genes and 135 downregulated genes. Figure 7 Parts A and C). Enrichment analysis revealed that the differentially expressed genes for the top 20 GO terms between the MIA group and the control group were mainly enriched in biological processes closely related to the progression of osteoarthritis, such as inflammatory response, chronic inflammatory response, and muscular system processes. Figure 7 (Part D). Previous studies have shown that elevated levels of pro-inflammatory cytokines disrupt the balance of the inflammatory system, leading to dysregulation of protein synthesis and degradation in muscle and joint cartilage, triggering synovial inflammation, and ultimately causing muscle atrophy, cartilage destruction, and pain. Furthermore, the US@BaTiO3-RVG@GA / HA group and the MIA group were enriched for sodium ion transmembrane transport-related GO terms (…). Figure 7 (Part E). Electromechanical coupling is considered to be the process by which VGSCs undergo conformational changes in response to membrane potential variations. This invention demonstrates that the local electric field generated by barium titanate nanoparticles under mechanical loading can directly inhibit channel opening and block Na+ by altering the conformation of transmembrane proteins. + Internal current, thus directly affecting the voltage sensing domain of VGSCs. Although Ca 2+ Modulation can block membrane receptor-mediated pain signal transduction, but its effects mainly occur in downstream pain transmission pathways, making it difficult to intervene in the initial electrical signals of peripheral sensitization. Furthermore, Ca2+ in nociceptive modulation... 2+ The signal may interact with Na through mechanisms such as sodium-calcium exchangers (NCX). +The invention modulates functional interactions. The results of this study suggest that the analgesic mechanism of analgesic microspheres may involve influencing sodium ion transmembrane transport to inhibit pain signal transduction, consistent with the inventors' hypothesis. Furthermore, gene set enrichment analysis of the inflammatory response (GSEA) confirmed that MIA can induce an OA inflammatory response and validated the successful establishment of the OA pain model in this study. Figure 7 (Part F). In the US@BaTiO3-RVG@GA / HA group, differentially expressed genes were mainly involved in inhibiting sodium ion transmembrane transport, as shown by GSEA analysis of this process. Figure 7 (Part G). KEGG analysis showed that genes significantly enriched between the MIA group and the control group were involved in the IL-17 signaling pathway, cAMP signaling pathway, and other pathways. Figure 15 (Part A). It is known that the IL-17 signaling pathway promotes articular cartilage inflammation and destruction by inducing the release of pro-inflammatory factors, increasing neutrophil chemotaxis, and stimulating the activity of synovial cell degradative enzymes. However, after treatment with analgesic microspheres, the inflammatory mediator regulation function of the TRP channel was enhanced (…). Figure 15 (Part B). TRPV channels, especially TRPV1, are involved in pain perception, inflammatory response, and structural damage in osteoarthritis. Their activation is associated with the release of inflammatory mediators, thereby exacerbating pain and inflammation. This invention found that TRPV1 expression in dorsal root ganglion (DRG) cells was significantly reduced after treatment with analgesic microspheres, which may be related to the internalization process of TRPV1 under the action of a piezoelectric field. In summary, RNA sequencing results further confirmed that the analgesic microspheres regulate the Nav1.7 channel through electrical pulses generated by piezoelectric biomaterials under the action of ultrasound, thereby inhibiting sodium ion influx, reducing pain signal transmission, and ultimately alleviating pain caused by osteoarthritis (OA).

[0085] 13. Histological evaluation

[0086] After treatment with analgesic microspheres, the rat knee joint tissue was separated and fixed in paraformaldehyde for one week. Images of the knee joint were then captured using a camera. Figure 16Joint tissues were decalcified with ethylenediaminetetraacetic acid (EDTA) (McKinlun, Shanghai) for 4 weeks, then embedded in paraffin and cut into 5-micrometer thick sections. Paraffin sections were stained with hematoxylin-eosin (H&E) and safranin O-fast green, and the pathological condition of the knee joint was assessed using a modified Mankin scoring system. After permeabilization and blocking, the tissue sections underwent immunohistochemical staining: first, they were incubated overnight with primary antibody for type II collagen (COL2) (1:300 dilution, catalog number AF0135, Anderson Biosciences) and secondary antibody for matrix metalloproteinase 13 (MMP13) (1:300 dilution, catalog number AF5355), followed by incubation with the secondary antibody for 1 hour, and then developed with 3,3′-diaminobenzidine (DAB) substrate. Finally, the expression levels of COL2 and MMP13 were quantitatively analyzed using ImageJ software.

[0087] Joint pathological features significantly influence osteoarthritis pain, but the relationship between pain and structural pathology remains unclear. This invention aims to investigate whether analgesic microspheres can further improve the joint pathological structure in osteoarthritis by relieving pain. Cartilage changes were assessed using H&E staining and Safranin O-Fast Green staining. Results showed that the control group had intact and smooth articular cartilage structure with no obvious damage and strong Safranin O-Fast Green staining; while the MIA, US, GA / HA, and BaTiO3-RVG@GA / HA groups showed significantly reduced articular cartilage matrix, severe surface erosion, and weaker staining. In contrast, the US@BaTiO3-RVG@GA / HA group showed reduced articular cartilage degeneration and relatively intact structure. Figure 8 Parts A and B). The Mankin-Kren system scoring showed that the injury index and total score were significantly reduced in the US@BaTiO3-RVG@GA / HA group. Figure 8 (Part D). Notably, no improvement in cartilage structure was observed in the BaTiO3-RVG@GA / HA group, indicating that ultrasound technology is crucial for piezoelectric analgesic microspheres. Furthermore, immunohistochemical analysis of COL2 and MMP13 protein levels showed increased COL2 expression and decreased MMP13 expression in the analgesic microsphere group compared to other groups. Figure 8Parts C, E, and F). Some scholars believe that MMPs (including MMP13) are members of the protease family and can decompose type II collagen and proteoglycans and hydrolyze chondroitin. The inventors’ research results show that analgesic microspheres may reduce cartilage degeneration by indirectly affecting chondrocyte metabolism. These microspheres can not only relieve osteoarthritis pain, but also reverse pathological changes. In a study on biodegradable injectable piezoelectric hydrogels made of polylactic acid (PLLA) and collagen matrix, the researchers found that ultrasound-mediated electrical stimulation can promote cartilage formation by inducing cell migration, promoting the release of transforming growth factor β (TGF-β), and upregulating the expression of chondrogenic genes such as COL2, aggregate proteoglycan (ACAN), and SOX9

[47] . In addition, some scholars designed a biodegradable piezoelectric nanofiber of poly-L-lactic acid (PLLA). They found that combining the piezoelectric scaffold with motion-induced joint load can successfully enhance in vitro cartilage differentiation and stem cell regeneration by attracting proteoglycan migration and promoting extracellular protein adsorption. These findings suggest that the role of analgesic microspheres in reversing joint pathological changes may be related to piezoelectric material-mediated endogenous TGF-β secretion and proteoglycan migration. While previous studies have revealed the potential of piezoelectric materials in promoting bone tissue repair, few studies have explored their modulatory effects on pain. This innovative study found that piezoelectric stimulation generated by analgesic microspheres not only achieves dual regulation of pain and cartilage repair but also effectively alleviates pain symptoms and inhibits disease progression in patients with osteoarthritis. However, further investigation is needed to clarify the specific mechanisms underlying these changes.

[0088] 14. Statistical Analysis

[0089] This invention uses GraphPad Prism 8.0 software for statistical analysis, and the results are expressed as mean ± standard deviation. To assess differences among multiple groups, one-way ANOVA and two-way repeated measures ANOVA were used. The significance level was set at P < 0.05.

Claims

1. A method for preparing targeted analgesic microspheres based on piezoelectric materials for treating osteoarthritis pain, characterized in that, The method includes the following steps: Rabies virus glycoprotein 29 peptide was grafted onto barium titanate particles and a BaTiO3-RVG solution was prepared. Then, a composite hydrogel made of GelMA and HAMA was placed in the aforementioned BaTiO3-RVG solution and freeze-dried under vacuum to obtain the analgesic microspheres.

2. The method for preparing targeted analgesic microspheres based on piezoelectric materials for treating osteoarthritis pain according to claim 1, characterized in that, The barium titanate particles are obtained by heat-treating a mixed solution of barium hydroxide, tetra(trimethylammonium chloride)titanium, titanium butoxide, ethanol and ammonium hydroxide at 200°C for 48 hours, followed by filtration, washing and drying.

3. A method for preparing targeted analgesic microspheres based on piezoelectric materials for treating osteoarthritis pain according to claim 1 or 2, characterized in that, When grafting rabies virus glycoprotein 29 peptide onto barium titanate particles, the rabies virus glycoprotein 29 peptide is dissolved in a mixed solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in a volume ratio of 5:3, then aminated barium titanate is added, and the reaction is stirred in an ice bath for 24 hours. The resulting product is purified by ultrafiltration.

4. The method for preparing targeted analgesic microspheres based on piezoelectric materials for treating osteoarthritis pain according to claim 3, characterized in that, The BaTiO3-RVG solution is obtained by placing barium titanate particles grafted with rabies virus glycoprotein 29 peptide in phosphate buffer.

5. The method for preparing targeted analgesic microspheres based on piezoelectric materials for treating osteoarthritis pain according to claim 1, characterized in that, In the composite hydrogel, the weight ratio of GelMA to HAMA is 5:

1.

6. The method for preparing targeted analgesic microspheres based on piezoelectric materials for treating osteoarthritis pain according to claim 5, characterized in that, The composite hydrogel was prepared using microfluidic technology.

7. The method for preparing targeted analgesic microspheres based on piezoelectric materials for treating osteoarthritis pain according to claim 6, characterized in that, In the microfluidic technology, the aqueous phase in the microfluidic system consists of 5 wt% GelMA, 1 wt% HAMA and 1 wt% photoinitiator, while the oil phase consists of 95 wt% paraffin and 5 wt% Span 80.

8. A method for preparing targeted analgesic microspheres for treating osteoarthritis pain based on piezoelectric materials according to claim 7, characterized in that, In the microfluidic technology, liquids of different phases are injected into a microfluidic device at an oil-water flow rate ratio of 1000 μL / min: 20 μL / min. After pre-gelling and collection, the liquids are rapidly frozen with dry ice and then transferred to a freezer at -80°C for overnight storage. After cross-linking with 405 nm ultraviolet light, the liquids are washed three times with acetone and deionized water to finally obtain the composite hydrogel.

9. A targeted analgesic microsphere based on piezoelectric material for treating osteoarthritis pain, characterized in that, The analgesic microspheres are prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the analgesic microspheres of claim 9 or the analgesic microspheres prepared by any one of claims 1 to 8 in the preparation of a medicament for treating osteoarthritis pain.

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